Rapid three-dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development
We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three-dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit-scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein-expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales.
© 2006 Wiley-Liss, Inc. Accepted 12 July 2006. Published online 18 August 2006. Grant sponsor: the National Institutes of Health; Grant number: 5R01HL078694; Grant sponsor: Swiss National Science Foundation; Grant number: PBEL2-104418; Grant number: PA002-111433 We thank S. Lin for the transgenic Tg(gata1:GFP) zebrafish line and Dimitris Beis and Didier Stainier for the Tg(tie2:GFP) line. We also thank Le Trinh and Liz Jones for helpful criticisms of these studies. S.E.F, M.E.D, and M.L. were funded by the National Institutes of Health, and M.L. was funded by the Swiss National Science Foundation.